JPH0335147A - Detection of chemiluminescence - Google Patents

Abstract

PURPOSE:To easily detect chemiluminescence by using a light emitting labeling compd. for detecting a slight amt. of nucleic acid or protein and quantitatively detecting the emitted light thereof as two-dimensional information. CONSTITUTION:The nucleic acid or protein which conjugates specifically with the specific nucleic acid or protein in a sample is labeled by either one of a chemiluminescent material or a material which catalyzes chemiluminescence and is specifically conjugated with the nucleic acid or protein in the sample. The chemiluminescence is thereafter effected in the presence of the catalyst or chemiluminescent material corresponding to the label thereof and the generated signal is detected by a photographic film, photomultiplier or photocounter. The chemiluminescent material is at least one kind of a luminol deriv., acrydinium deriv., etc., and the catalyst is a metal complex having a porphyrin deriv. structure. The metal complex contains at least one kind of iron, cobalt, manganese, nickel, chromium, zinc, osmium, molybdenum, cadimium and copper.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel microanalysis method for nucleic acids and proteins using chemiluminescence.

Research at the genetic level in molecular biology has made great progress with the discovery of restriction enzymes, the development of various vectors, and advances in cell culture technology. Among these, techniques that use probes to detect fragments of nucleic acids with specific sequences, such as the signature method, the Northern method, colony hybridization, and plaque hybridization, are indispensable for advancing research. Furthermore, ELISA and immunoassay using monoclonal antibodies are also routinely performed.

2 Conventionally, these detection methods use radioactive isotopes such as P, 35S, or However, there are problems with the danger to the human body and the environment and the instability of the obtained labeled substances, and the 1 m of labeling methods that do not use radioactive isotopes. The outbreak is being actively investigated.

Systems have already been devised in which a fluorescent dye is introduced, or a zero element such as peroxidase or alkaline phosphatase is introduced, and detection is performed using an oxidative coloring reagent or a chemiluminescent reagent. However, these systems lack sufficient sensitivity and are complicated to operate compared to those using conventional radioisotopes. Alternatively, there is room for improvement in that the properties of the label may change by adding a compound with a large molecular weight such as zero element.

5+-3 Volume i°rut・Meno-2゛For the above reasons, the present inventors used chemiluminescent substances with small molecular weight or chemical Considering that if a compound that catalyzes luminescence is used as a labeling compound, it would be possible to more selectively detect where on the planar g4rMm body the compound recognized by the labeling compound exists. As a result of research on detection methods for luminescent labeled compounds, the present invention was completed. That is, the present invention is a method of using a luminescent labeling compound to detect trace amounts of nucleic acids or proteins, and quantitatively detecting the luminescence as two-dimensional information. Photographic film is used as a detection method.

For example, chemiluminescence can be easily detected by using an instant film. Furthermore, by counting chemiluminescence using a photon counter, the amount of light emitted by the same device can be measured, and by using a multi-photon counter, information regarding the location of the light emission can be obtained.

By using a compound that recognizes a nucleic acid or protein with a molecular weight smaller than that of an M element, the recognition ability of the labeled substance is greatly improved.

As a chemiluminescent system, for example, the following means can be used.

1) A 'o-phenanthroline derivative and hydrogen peroxide are reacted in the presence of a copper (n) or osmium (■) catalyst to emit light.

2) A luminol derivative and hydrogen peroxide are reacted with a metal such as iron, cobalt, manganese, nickel, chromium, zinc, osmium, molybdenum, cadmium, or copper or a complex thereof as a catalyst to emit light.

3) React the acridinium derivative and hydrogen peroxide to emit light.

4) React the oxalate ester derivative with hydrogen peroxide in the presence of a fluorescent 'rIJ substance to emit light.

Among these, in 1) and 2), the reaction proceeds due to the catalytic action of trace amounts of metal ions, so the target compound may be labeled with the substrate phenanthroline or a luminol derivative, or the catalytic metal ion may be used as a chelate compound to target the target compound. The compound may be labeled, 3) is a non-catalytic reaction and must be labeled with an acridinium derivative, and 4> may be labeled with an oxalate ester derivative, or tlIwA with a fluorescent substance. A good labeling method is to label the chemiluminescent substance or catalyst with an amino group, carboxyl group,
This is carried out by using a compound into which a functional group such as a formyl group, azide group, or inthiocyanate group has been introduced, and reacting it with a nucleic acid or protein via the functional group or using a bifunctional reagent.

In addition, as a method for detecting the generated chemiluminescence, a-sensitive instant film such as Polaroid Type 612 film (ASA 20°000) or high-sensitivity It is brought into contact with a cellulose filter or the like through a thin plastic or glass plate, and a solution containing hydrogen peroxide and a catalyst or chemiluminescent substance is applied onto the gel plate or filter to cause it to emit light and be exposed to light. Alternatively, similar results can be obtained by emitting light from the labeled compound on the plate using the above method in a dark room, and then using a camera loaded with high-sensitivity film to obtain the same results. It may be detected.

Specific methods for modifying target compounds with these labeling compounds and detecting them two-dimensionally include α, β, and γ.

By reacting δ-tetrakis(4-aminoethylcarbamoylphenyl)porfinato Fe (III) with the amino group of the nucleobase of an oligonucleotide having a specific base sequence via glutaraldehyde, the nucleic acid is converted to Fe.
(II) Label with a complex catalyst and use as a probe. This is done using the conventional method (T, ManLaLis.

et al,'Mo1ecular Clonin
g A Laboratory Manual”
, Colorado Spring Harbor La
D.
The probe of the nitrocellulose filter onto which the NA or RNA fragment has been transferred is hybridized with a fragment having a complementary sequence, and the probe is brought into close contact with a Polaroid instant film and a thin plastic plate. Next, open the shutter cover of the instant film set 1~ in the darkroom,
An alkaline aqueous solution of hydrogen peroxide and luminol is added to a nitrocellulose filter to generate chemiluminescence. After about ten minutes of exposure, the film is removed from the holder and developed, allowing the fragments having complementary sequences to the probe to be detected as white bands. By using such chemiluminescent labels and photography, two-dimensional detection of trace amounts of substances can be performed safely and quickly, without using radioisotope labels, which are cumbersome to handle and take time to obtain results. be able to do it.

In order to find out the detection limit of the above chemiluminescence system, the detection sensitivity of chemiluminescence was measured using a simple system. For systems using 0-phenanthroline, CEDAB (cationic surfactant)
1. OXIO-2M, sodium hydroxide 3.5x10-
2M14(II)4.3XIO-4M and hydrogen peroxide 3
．． When the total volume of the solution is 35111 at 0%, luminescence can be detected by photography up to 0-phenanthroline 2.5X10''-5Mf).Also, osmium (■) 6.7810-8M instead of copper(II) Similar detection sensitivity is obtained using .

Next, in systems using luminol derivatives, α.

β, γ, δ-tetrakis(4-carboxyphenyl>porfinato Fe(I[[) (hereinafter abbreviated as TCPP-Fe)) 5.OXlo-8M and hydrogen peroxide 0.03%
If the total volume of solution is 30 μl, Luminol 1. OXl
Can be detected at o-7M. In addition, α, β, γ, δ-tetrakis(4-aminoethylcarbamoylphenyl)porfinato Fe (■) (hereinafter abbreviated as TCPPA-Fe)
If this is done using a method of labeling using glutaraldehyde, Luminol 1. OXIO-5M, hydrogen peroxide 0.
When the total volume of solution is 30 μl at 0.3%, TCPPA-F
It was found that even astringent skin of e6.7X10-14M could be detected.

The detection limit in the system using acridinium derivatives is 4-(
Proteins were labeled using 2-succinimidyloxypolonylethyl)phenyl-10-methylacridinium-9-carboxylate fluorosulfonate, and 0.
03% hydrogen peroxide.

Total ff121 using 20mM sodium hydroxide as luminescent liquid
When performed at 400, ul, it was confirmed to be 8.0×10 M.

Although the outline of the present invention has been described above, the chemiluminescent reaction in the present invention is not limited to the above four types,
It can be applied to all labelable substrates and catalyst systems. Also, the film used for photography is Polaroid 61
Films with sensitivity applicable to the purpose of the present invention, including but not limited to 2 films and high-sensitivity X-ray films,
It also includes photoreceptors such as photographic paper. At the same time, photon detection systems such as photon counters are also included in this application range.

X-Benefit 1 The content of the present invention will be explained in more detail with reference to Examples below.

Example 1 A method of detecting DNA Alli fragments having a complementary sequence by photographic method using DNA labeled with a luminol derivative as 10-label l) Dot blotting on a nitrocellulose filter Heat treatment on a 2% 3 cm nitrocellulose filter single-stranded λ phage DNA, lOpg, 1100p
, lng, 10% g and 10011 g with a diameter of 4 mm
It was spotted to the size of , and baked at 80° C. under reduced pressure for 2 hours.

2) Creation of DNA probe 10% g of λ phage DNA (cleaved with Hindl [I) was made into a single strand by heat treatment, and then 10 μl of 0.3M N-(4-aminobutyl)-N-ethylisoluminol/DMSO solution was added. and 1 μm of 5% glutaraldehyde. The total volume was adjusted to 50μ■, and the reaction was carried out for 10 minutes at 37°C.
The reaction product was purified by ethanol precipitation.

3) Hybridization on a nitrocellulose filter The nitrocellulose filter prepared in step 1 was placed in a heat-sealed bag and prehybridized for 2 hours at 42°C, then a DNA probe was added and hybridized for 16 hours. Ta. The nitrocellulose filter was washed three times with water.

Hybridization was carried out using a conventional method (T, Mania
Lis, eL al, “Mo1ecular Cl
oning A Laboratory Man
ua+”, Co1d Spring Harbor
Laboratory, 1982).

4) Detection of chemiluminescence by photography A container with a plastic film on the bottom was placed on top of a Polaroid 612 instant film cassette, and a luminescent solution (0.03% hydrogen peroxide, 5.0
X10-8M-TCPP-Fc aqueous solution, 3. OXlo
-2M-carousic acid, and 6. After soaking the nitrocellulose filter in OXIO-2M (potassium hydroxide),
It was placed on a plastic film and exposed to light for 10 minutes in a dark room with the shutter cover open. As a result of removing the photographic film and checking the luminescence, it was possible to detect up to 10% g spots.

Example 2 Adding a porphyrin iron derivative to DNA via a spacer
Method for detecting DNA fragments with complementary sequences using a photographic method using identified probes 1) Preparation of probe 50 rng of TCPP-Fel was mixed with 3.1 g of WSC<water-soluble carbodiimide) and 2.1 g of ethylenediamine.
g in a THF/H20 mixed solvent, α, β, γ,
δ-tetrakis(4-aminoethyl!levamoylphenyl)porfinate Fe(III) was developed. Next, 8 μl of a 2.4×10 −4 M aqueous solution of this compound,
9.4k isolated by cutting λ phage DNA with Hind.
bf) The reaction product obtained by reacting 5 μg of the DNA fragment with 3 μl of 0.5% glutaraldehyde was also subjected to ethanol precipitation.

2) Southern blotting onto a nitrocellulose filter Lambda phage DNA was cut with Hindl and loaded on a 0.8% agarose gel at 0.1, 0.5 and 1.0% g for electrophoresis. Then, the agarose gel was
The DNA was immersed in an aqueous NaCl solution to obtain single-stranded DNA, neutralized, and transferred to a nitrocellulose filter. After baking the filter, it was placed in a heat seal bag and pre-piperidized at 42°C for 2 hours.
10-b was added and piperidized for 16 hours. After piperidization, the nitrocellulose filter was taken out and washed with water.

3) Detection of chemiluminescence by photographic method Luminescent liquid (0.01% hydrogen peroxide, 3.4X10-2M luminol, 3.OXIO-2M boric acid, 6.0XIO")
Luminescence was detected in the same manner as in Example 1 using -2MKOH). As a result, l, 10.1100n amount of DNA
In both cases, only the band b was detected at 9.4.

Example 3 Using a monochrome Nano 1 antibody labeled with an acridinium derivative as a probe, a DN encoding the protein of interest was prepared.
Method for detecting A 1> aK recognition of monoclonal antibody A monoclonal antibody that recognizes the C-terminal side of 10 thioglycan of rat cancer JLL cell line L2 as an epitope,
4-(2-Succinimidyloxycarbonylethyl)phenyl-1o-methylacridinium-9-carboxylate was reacted with fluorosulfone & 2 salt and vA was identified.

2) Transfer of hybrid protein to nitrocellulose filter Using a DNA library prepared using rat RBLI, It cell strain λ, 11, Eco
1Y1090 to form plaques on 20 agar plates, and then transferred to nitrocellulose filters. At this time, the number of plaques is 1.0XIO4
The samples were prepared in such a way that the number of cells/plate was obtained. The filter was placed on an agar plate containing isopropyl-β and D-galactose and cultured. The filter was washed with PB at pH 7.4.
After immersing in a mixture of SKI solution and acridinium-labeled monoclonal antibody for 2 hours, it was washed with PBS Enomai solution.

3) Detection of chemiluminescence by photographic method A nitrocellulose filter was immersed in a luminescent solution (0.03% hydrogen peroxide, 20 mM sodium hydroxide).
Chemiluminescence generated by the same procedure as above was detected. As a result, two spots were detected from 20 filters. These subofts were found to be included in the spots detected by screening using DNA encoding the L2 proteoglycan core protein as a 10-propane.

Example 4 Method for photographically detecting proteins labeled with porphyrin iron derivatives 1) Synthesis of porphyrin iron (m) derivatives Carbon disulfide 10
0 rn g and 1 ml of triethylamine were combined with flj,
To this, α and β were synthesized in 'A8i12.

1 m of γ, δ-tetrakis(4-aminoethylcarbamoylphenyl)porfinato Fe(Ift) Loomg
Dissolve in 1 of THF and add at O''C.

β, Fu, δ-tetrakis(4-inthiocyanatoethylcarbamoylphenyl)porfinatoFe(III)
(TCPPI-Fe) was synthesized.

2) Due to protein porphyrin iron derivatives! Phosphorus M at pH 7.2 containing 5 mg of human serum albumin (H3A)
[1 ml of W solution, 1 mg of TCPPI-Fe/1 DMSO]
(The SA-labeled 0 reaction mixture was dissolved in 1 ml of phosphoric acid M solution purified using Sephadex G50.)

3) Detection of chemiluminescence by photography 966 microtiter 7 rates, lpg,
i 0pg, 1100p, lng, 10ng, loon
gf) Add the above solution so that it contains fi-specific H3A,
Make sure the total amount is 100μX.

Phosphoric acid [fl? liquid was added. Next, 100 μm of the luminescent liquid used in Example 1'A2 was added, and the chemiluminescence produced was transferred onto a film in the same manner as in Example 1. The exposure time was 10 minutes. As a result, 1 pg of labeled HSA could be detected.

4) Detection of chemiluminescence using a photon counter Using a microtiter plate in the same manner as in 3), add labeled H3A from lpg to 1100n.

The total amount was 100 μm, and the luminescent liquid used in Example 2 was 100 μm.
μm was added and the resulting chemiluminescence was counted. As a result, it was found that the labeled HS Affi could be sufficiently detected even in lpg.

that's all

Claims (1)

[Claims] 1) In a method for detecting a specific nucleic acid or protein in a sample, the nucleic acid or protein that specifically binds to the nucleic acid or protein is replaced with either a chemiluminescent substance or a substance that catalyzes chemiluminescence. Detection characterized by labeling and binding specifically to the nucleic acid or protein in the above-mentioned material, then performing chemiluminescence in the presence of a catalyst or chemiluminescent substance corresponding to the label, and detecting the resulting signal. Method. 2) The detection method according to claim 1, wherein the chemiluminescent substance is at least one substance selected from the group consisting of luminol derivatives, acridinium derivatives, o-phenanthroline derivatives, and oxalate ester derivatives. 3) The detection method according to claim 1, wherein the catalyst is a metal complex having a porphyrin derivative structure. 4) The metal complex is iron, cobalt, manganese, nickel,
Chromium, zinc, osmium, molybdenum, cadmium,
The detection method according to claim 3, wherein the detection method is a metal complex containing at least one metal selected from the group of copper. 5) The method for detecting the luminescent signal is a photosensitive photographic film,
The detection method according to any one of claims 1 to 4, characterized in that a photomultiplier or a photon counter is used.